論文

We present an experimental system for real-time monitoring of 3D constructed tissues at single-cell resolution to further understand cellular response against mechanical stimuli. The system enables us to continuously monitor the same cells in a 3D tissue at a subcellular level under uniaxial cyclic stretch. We confirmed our system's effectiveness by observing that 3D tissues cultured under cyclic stretch were oriented to the direction of stretch. Moreover, cells in a 3D tissue were successfully observed continuously at single-cell resolution even under cyclic stretch. We believe that the real-time observation of cellular response against mechanical stimuli with our demonstrated system could be an effective method for investigating the relationship between cellular response and mechanical stimuli in detail.

Surgery, radiation therapy, and chemical therapy have been reported as main treatments for cancer that is one of the worst diseases reported. However, because of high invasiveness on patients, cancer hyperthermia has been studied as non-invasive treatment. Hyperthermia uses a difference of thermal tolerance between normal and cancer cells and gives an affected part thermal stimulation to kill cancer cells selectively. For developing effective conditions of hyperthermia, in vitro study to evaluate the thermal tolerance have been required. However, since existing cell culture vessels cannot control the culture temperature, genuine thermal tolerance of cells cannot be investigated appropriately. To reveal critical temperature proper for hyperthermia, we developed a culture device controlling culture temperature. With the developed device, culture temperature was regulated far more quickly than conventional method with existing vessels, which enable us to study the thermal tolerance of cells appropriately. For the control of culture temperature, the device has a titanium culture substrate where a peltier element adhered. Since a biocompatibility of the device was confirmed, a difference of thermal tolerance between normal and cancer cells was, then, investigated with the developed device by using Normal Human Dermal Fibroblasts and Michigan Cancer Foundation-7 as model cell species. As a result, it was confirmed that cancer cells were more sensitive to thermal stimulation than normal cells, which was qualitatively consistent with previous researches. Thus, we conclude our developed device can be used for investigation of thermal tolerance of each cell specie, which will contribute for the development of cancer hyperthermia.

Biomimetic scaffolds are required in tissue engineering to provide structural support as well as promote cell adhesion, proliferation, and differentiation. Fibrous scaffolds composed of micro- and nanofibers replicate the architecture of the native extracellular matrix. Electrospinning is widely used for fabricating nanofibers; however, constructing fibrous scaffolds that integrate multiple fiber scales into a single structure is difficult. We addressed this issue by developing a fiber-spinning device using two pairs of counter-facing syringes that simultaneously produce micro- and nanofibers under different processing conditions. Poly(ε-caprolactone) solutions are ejected through needle-type nozzles via centrifugal force, and fiber diameter is controlled by adjusting the polymer concentration and nozzle diameter. We fabricated scaffolds with the proposed device, which exhibited a random three-dimensional fibrous network in which microfibers and nanofibers were homogeneously integrated. C2C12 myoblasts cultured on the composite scaffolds strongly adhered to the fibrous network, remained viable, and extended along the fibers to form multinucleated cells within the structure. The developed system produced composite micro/nanofiber scaffolds with tunable morphology and biocompatibility, providing a platform for fibrous tissue engineering applications.

Cell analysis is vital in clinical diagnostics and cell engineering research. Among the various analytical techniques, dielectrophoresis (DEP) is a particularly promising label-free method for distinguishing biological particles, which eliminates the need for fluorescent dyes or magnetic beads. In this study, we present a high-precision single-cell analysis system based on the evaluation of DEP forces in a controlled microfluidic flow environment. The system integrates a microfluidic chamber equipped with an electrode array to exert DEP forces and flow-induced shear forces to facilitate force balance analysis. We quantitatively characterized the DEP response to successfully discriminate between healthy skin cells and cancer cells using the proposed DEP-based cell-sorting platform. The proposed system successfully distinguished between these cell types even when their dielectrophoretic properties were similar, highlighting its potential for sensitive and selective cell classification in biomedical applications.

Mechanomedicine represents a potential biocompatible method in cancer therapy. In particular, the use of low-frequency mechanical vibration previously proved to trigger apoptosis of the human epidermoid carcinoma A431 cell line. In this study, we further characterized the metabolic and oxidative stress responses triggered by 1 h of 20 Hz mechanical vibration stimulus to A431 prior to cell death. Our results indicate that cell death may be related to the decrease of glucose consumption rate and the higher expression of reactive oxygen species right after mechanical stimulation (0 h). The overexpression of HMGB1 and HSP70 coding genes signified the increase of A431 cell stress. However, HMGB1 and HSP70 expression decreased at 24 h after mechanical vibration, along with the progression of cell death. We also observed cell morphology changes on A431 cells following vibration which might be indicative of A431 death by apoptosis. The emergence of these stress responses suggests that several pathways are connected to promote cancer cell death. The discovery of A431 cellular stress symptoms which lead to apoptotic death may clarify the usefulness of mechanical vibration in cancer treatment as a novel application of biomechanical manipulation.

Pluripotent stem cells (PSCs) such as embryonic stem cells and induced PSCs can differentiate into all somatic cell types such as cardiomyocytes, nerve cells, and chondrocytes. However, PSCs can easily lose their pluripotency if the culture process is disturbed. Therefore, cell sorting methods for purifying PSCs with pluripotency are important for the establishment and expansion of PSCs. In this study, we focused on dielectrophoresis (DEP) to separate cells without fluorescent dyes or magnetic antibodies. The goal of this study was to establish a cell sorting method for the purification of PSCs based on their pluripotency using DEP and a flow control system. The dielectrophoretic properties of mouse embryonic stem cells (mESCs) with and without pluripotency were evaluated in detail, and mESCs exhibited varying frequency dependencies in the DEP response. Based on the variance in DEP properties, mixed cell suspensions of mESCs can be separated according to their pluripotency with an efficacy of approximately 90%.

In this study, we proposed an in vitro tumor model to simulate the mechanical microenvironment and investigate the effect of compressive stress on the invasion process of malignant tumors. It has been pointed out that the biomechanical environment, as well as the biochemical environment, could affect the transformation of cancer cell migration, invasion, and metastasis. We hypothesized that the solid stress caused by the exclusion of surrounding tissue could transform tumor cells from noninvasive to invasive phenotypes. Colorectal cell spheroids were embedded and cultured in agarose gels of varying concentrations to simulate the earliest stages of tumor formation and invasion. The spheroids embedded in gels at higher concentrations showed peculiar growth after 72 h of culture, and the external compressive loading imposed on them caused peculiar growth even in the gels at lower concentrations. In conclusion, the mechanical microenvironment caused the transformation of tumor cell phenotypes, promoting the growth and invasion of tumor cell spheroids.

Induced pluripotent stem cells (iPSCs) are a promising cell source because of their pluripotency and self-renewal abilities. However, there is a risk of pluripotency loss during cell expansion. Particularly, cell passaging is associated with a higher risk of decreasing cell quality. There are two iPSC passaging methods: single-cell and clump passaging. Single-cell passaging is a rapid and simple method for cell manipulation, whereas clump passaging is superior for maintaining iPSC pluripotency. Therefore, clump passaging is a robust method for expanding iPSCs while maintaining their pluripotency. However, clump size control during clump passaging is difficult because colony fragmentation is performed manually by pipetting the colonies detached from the cell culture substrates. In this study, the effect of pipetting on iPSC colony fragmentation was evaluated and the relationship between iPSC clump size and pluripotency was clarified. An automated pipetting device was developed to standardize the clump passage process. The effect of clump size on the pluripotency and proliferative capacity of mouse iPSCs was investigated. Clump size was controlled by varying the number of pipetting cycles, and pluripotency and proliferation were assessed via alkaline phosphatase staining and flow cytometry. Our results revealed that a decrease in clump size corresponded to an increase in cell proliferation, while pluripotency maintenance was optimized under specific clump sizes. These results underscore the significance of clump size for stem cell quality, emphasizing the need for a balanced approach to maintain pluripotency while fostering proliferation in the cell expansion culture for iPSCs.

Various culture devices have been developed as fundamental technologies for facilitating bioengineering studies. Culture devices are designed to prepare specific culture environments. Thus, both macrostructures and surface micromorphology should be considered in the device design. Although fabricating devices with elaborate designs incurs high production costs, disposable materials are typically used for culture devices. However, some metallic materials are strong, stable, and biocompatible. Bioengineers have not applied these materials to culture devices because of the difficulty of processing. An emerging technology using three-dimensional (3D) printing has been developed, which can produce complex designs using metal. We demonstrate the applicability and potential of metal 3D printing for fabricating culture devices toward the development of the bioengineering discipline. As a specific example, we fabricated metallic culture devices where the environment of cultured tissues can be improved. One of the biggest factors determining the culture environment is active media supply. To attain active media supply to the tissue, devices having culture surfaces with mesh structures having holes far larger than cells were proposed. Cell sheets were cultured as tissue models, realizing tissue culture with such structures. The cultured tissue showed increased metabolism, indicating enhanced media supply owing to mesh surfaces. The biocompatibility of the 3D printed metal device was confirmed by viability assays on cultured cells, and reusability of the device was confirmed by mechanical and biochemical evaluations. We believe this study serves as a reference for using metallic 3D printing as an option for fabricating culture devices, which will promote bioengineering research.

Background: Platelets contain cytokines and growth factors, which promote wound healing and tissue regeneration. Platelet concentrates, known as platelet-rich plasma (PRP), are used for tissue repair and regeneration in clinical settings. PRP is conventionally prepared by the centrifugation of blood; therefore, blood cells such as erythrocytes and leukocytes cannot be fully eliminated. The purpose of this study was to establish high purification of platelets using dielectrophoresis (DEP) to eliminate blood cells from platelet concentrates. Materials and Methods: The DEP properties of human blood constituents (erythrocytes, leukocytes, and platelets) were characterized. Based on the characterization of DEP properties, PRP was purified to eliminate blood cells using dielectrophoretic and flow-induced shear forces. Results: Erythrocytes and platelets showed similar DEP responses, whereas the crossover frequency of leukocytes differed from those of erythrocytes and platelets. Blood cells can be eliminated from the PRP under specific DEP conditions and flow rates. Conclusion: Blood cells in PRP can be eliminated using DEP and flow-induced shear, with a high platelet recovery rate.